Light microscopy is based on propagating light from an illuminated sample which is passed through a set of lenses resulting in an enlarged view of the desired object. This basic principle is used in a variety of microscopic techniques but suffers from a range of deficiencies such as limited resolution and reduced image clarity. The optical resolution in light microscopy is due to diffraction of light and therefore objects smaller than 250 nm are difficult to resolve. This is even worse in the z direction (optic axis), where this limit is extended to 500 nm or more. Nevertheless, many cellular structures and components are often smaller than this optical resolution limit and determining the properties of biomolecules such as proteins in their natural environment is important when analysing their function and elucidating cellular processes. The application of microscopes in life and material science is ever increasing and methods allowing the imaging of small objects under physiologically conditions are highly desirable. Resolution in live samples is generally lower than that in fixed specimens because of the size of the sample, the scattering of tissue, lack of pigmentation and the movement of cellular components.
Illumination techniques such as STimulated Emission Depletion (STED) microscopy, Structured Illumination Microscopy (SIM), or single-molecule-based (SM) techniques (PALM/STORM) have revolutionised microscopy and enabled so called ultra-high resolution. Although these techniques offer clear advantages in terms of spatial resolution over the traditional illumination methods, creation of these images require complex instrumentation and data analysis. These techniques suffer from deep imaging capabilities of live biological samples.
Fluorescence Light-Sheet microscopy techniques have become increasingly popular and are more suitable for imaging live cells. The idea behind light-sheet-based microscopy techniques is to illuminate only a thin layer of the sample from the side, vertical to the direction of observation in a well-defined volume around the focal plane of the detection optics. This technique does not require the use of strong lasers making it minimal invasive and reducing photobleaching.
In a widely adopted light-sheet technique Selective Plane Illumination Microscopy (SPIM) cylindrical optics or scanning through galvanometric mirrors are used to create a sheet of light of varying thickness and can be adapted to different sample sizes: for smaller samples (20-100 μm), the light-sheet can be made very thin (˜1 μm), whereas for larger samples (1-5 mm), the sheet has to be thicker (˜5-10 μm) to remain relatively uniform across the field of view.
In contrast to the detection system used in epifluorescence microscopy, where a single objective lens is used to both illuminate the sample and to collect its fluorescence along the same path, SPIM comprises: (1) a detection lens horizontally aligned and immersed in a fluid-filled chamber, with a sample embedded in a transparent gel and immersed in the chamber medium held from the top; (2) an excitation lens to illuminate the sample perpendicularly to the optical axis of the detection lens; and (3) single cylindrical lens, or galvanometric mirrors, forming the light-sheet inside the chamber through the excitation lens. A stack of images is acquired by moving the sample in a stepwise fashion along the detection axis.
Although Fluorescence Light-Sheet microscopy addresses in principle some of the limitations encountered by other techniques, complex machinery and difficult set ups make this method unsuitable for routine laboratory practise. As described above, Fluorescence Light-Sheet microscopy requires 2 objectives to be placed perpendicularly and close to the sample, which besides the distinctive machinery requires also special sample holders and prevents using high NA objective and regular coverslips. It is apparent that there is no optimal solution which can address the issue of imaging in 3D an entire single cell with best possible nanometric resolution provided by SM-based super-resolution microscopy.
This disclosure relates to a device for containing a sample which is adapted to fit to any microscope assembly and provide light sheet microscopy of a sample or samples contained in the device. The device includes a sample well wherein one or more sides of the well are provided with an angled reflective surface adapted to reflect a light sheet transversely through a sample to provide a fluorescence image detectable by a single objective. The light sheet and fluorescence collection are performed through the same objective. The device provides a simplified and inexpensive solution to the aforesaid problems associated with high resolution fluorescence microscopy. The disclosure provides a single objective SPIM [soSPIM] approach and allows performing SPIM imaging on a standard inverted microscope by virtue of an array micro-mirrored chip. The detection and excitations are performed through the same and unique single objective. The device can be scaled to include variable size reflective surfaces (e.g. from 20 microns to 2 mm) and using the appropriate magnification objectives (e.g. from 100× to 10×), the soSPIM system allows 3D SPIM from 3D high- and super-resolution of a single cell, up to the whole organism level, [for example embryo imaging], on the same instrument.
The disclosure demonstrates 3D imaging capabilities using 100×, 60×, 40× 20× and 10× objectives with excellent resolution and SM-based super-resolution microscopy. Advantageously, 3D optical sectioning using the device does not require moving the sample, but only the objective and the light sheet, allowing acquisition speeds comparable with other imaging techniques such as spinning-disc microscopy.
Moreover, the use of arrayed devices allows simultaneous imaging of multiple cells. This provides the capability to image multiple single cells simultaneously to dramatically reduce the acquisition time and improve imaging throughput. The arrayed devices can contain thousands of single cell wells facilitating sample processing of cells and even whole organisms, such as embryos.